LOCUS       sequence-388752-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For affinity purification of Pnf payload.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7106)
  AUTHORS   Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F
  TITLE     Programmable protein delivery with a bacterial contractile injection
            system.
  JOURNAL   Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7.
            Epub 2023 Mar 29.
  PUBMED    36991127
REFERENCE   2  (bases 1 to 7106)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-023-05870-7"; journalName: "Nature"; date: 
            "2023-04"; volume: "616"; issue: "7956"; pages: "357-364"
COMMENT     Sequence Label: TwinStrep-SUMO-Pnf
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(7..12,13..29,30..35)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(210..257)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     253..271
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1344..1637)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(join(1638..1661,1662..1697,1698..1721))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(1734..1751)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1761..1778)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1797..1819
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1834..1858
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(1858..1877)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1859..1877)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1961..1980)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2186..2263
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2264..3346
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2283..2302)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4658..4849
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4843..4865)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4951..5090
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5006..5025
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5105..5122)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5276..5864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5356..5375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6035..6826,6827..6895))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6658..6677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6896..7000)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7068..7086
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcatgtttga cagcttatca tcgataagct ttaatgcggt agtttatcac agttaaattg
       61 ctaacgcagt caggcaccgt gtatgaaatc taacaatgcg ctcatcgtca tcctcggcac
      121 cgtcaccctg gatgctgtag gcataggctt ggttatgccg gtactgccgg gcctcttgcg
      181 ggatatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
      241 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg
      301 gcctcgagtg cggccgctta taacaaccgt tttttaagtg aattaatggc cttaacagta
      361 tttccctgct tacctgatga aacgataaca tcttccgata gtaccttaac gttcaccttt
      421 cctttatcgt ccttagccag caacgcataa ctgtagccag ctctaatagc aggtttttca
      481 ggcttaattt cgtcataatt aaaaacacca acattttctg ccattttatc aatcagcact
      541 cccggttttc ccatataggt aataacactc tgatcataat ttgcaaaaat attcactaaa
      601 tcgttattct gttcaccaac cgtcataggt tcactatccg gcgaaagggc ttgatggctc
      661 agataggtag caacaacacc ctgcctgcct gttttccatt cattattacc cggtttctgc
      721 cccgtatgat aggcaaaaaa atagccattt ttcaccgaat ataccatagt acaaccgctc
      781 aatgctccag acgtgacaat aaccggggac ccatgctgaa tatccttcaa agcaattctt
      841 attcccacgg ttcccgatgc accattaacc acattaacac ccataatatt ggaatccagc
      901 gtatttttcc cccaatgtac gcctattgat cctgaacccg actctgatga tgtcacctga
      961 tcaccacgaa atcctgcaat aacaccataa aaagggtcta gatgattgga gtgactatca
     1021 gctttcaaag gctccgcatc tgtaagttga tggctgctgt cataacgatg agctataact
     1081 gggatgtcaa ttgacacatc cttactacct actaataatc ccttattcat caacggggaa
     1141 atatgccctt tattaagact cctatccgca agatgttgtc tcaatccttt gattttccta
     1201 atcttatggc cttcgtaagg ctggttttca ccatttgaaa gttcaaggtg cccatcaaaa
     1261 gaggttgatg atggcggttt cttcgcagta tttttagtac gttgtgtggc tacggtctga
     1321 ggattagcat attttaacat ggatccacca atctgttctc tgtgagcctc aataatatcg
     1381 ttatcctcca tgtccaaatc ttcaggggtc tgatcagctt ggattctaat accgtcgtac
     1441 aagaatctta aggagtccat ttccttaccc tgtcttttag cgaacgcttc catcagcctt
     1501 cttaaaggag tggtcttttt gatcttgaag aagatctctg aagatccatc ggacaccttt
     1561 aaattgatgt gagtctcagg cttgacttct ggcttgacct ctggcttagc ttcttgattg
     1621 acttctgagt ccgacatttt ctcaaactga gggtgtgacc atgcactacc acctgaacca
     1681 ccaccgctac caccaccttt ttcaaactgc ggatggctcc agctagccat atggctgccg
     1741 cgcggcacca ggccgctgct gtgatgatga tgatgatggc tgctgcccat ggtatatctc
     1801 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     1861 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
     1921 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     1981 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     2041 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     2101 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     2161 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     2221 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     2281 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     2341 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     2401 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     2461 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     2521 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     2581 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     2641 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     2701 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     2761 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     2821 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     2881 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     2941 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     3001 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     3061 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     3121 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     3181 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     3241 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     3301 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     3361 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
     3421 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     3481 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     3541 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     3601 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     3661 aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga
     3721 cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc
     3781 ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag
     3841 gatcgctcgc ggctcttacc agcctaactt cgatcattgg accgctgatc gtcacggcga
     3901 tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat
     3961 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa
     4021 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt
     4081 cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc
     4141 catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg
     4201 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     4261 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     4321 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     4381 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     4441 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     4501 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     4561 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     4621 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     4681 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     4741 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     4801 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     4861 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     4921 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     4981 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5041 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     5101 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5161 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5221 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5281 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5341 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5401 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5461 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5521 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5581 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5641 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5701 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5761 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5821 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5881 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5941 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6001 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6061 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6121 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6181 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6241 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6301 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg
     6361 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6421 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6481 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6541 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6601 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     6661 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6721 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6781 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6841 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6901 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6961 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7021 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7081 cgaggccctt tcgtcttcaa gaattc
//
